Interleukin 1-dependent induction of both interleukin 2 secretion and interleukin 2 receptor expression by thymoma cells

The macrophage-derived product, interleukin 1 (IL 1) is thought to play an important regulatory role in the proliferation of T lymphocytes; however, its mechanism of action is unknown. We describe in this report a variant subline of EL4 thymoma cells (EL4-6.1) that displays a high degree of responsiveness to IL 1. We show that recombinant IL 1 can induce both the secretion of interleukin 2 (IL 2) and the expression of IL 2 receptors (IL 2-R) by these cells. EL4-6.1 cells do not constitutively secrete IL 2, nor do they express IL 2-R; but when cultured in the presence of recombinant IL 1, they secrete detectable amounts of IL 2 (5 to 15 U/ml). In the presence of either suboptimal levels of phorbol ester (PMA) or Ionomycin, the addition of IL 1 resulted in up to an 80-fold enhancement in the amount of IL 2 secreted. Stimulation with IL 1 alone or in combination with Ionomycin was unable to induce detectable IL 2-R expression by EL4-6.1 cells. However, in the presence of suboptimal concentrations of PMA, IL 1 induced expression of about 3000 high affinity (dissociation constant, Kd of 31 pM) and 50,000 low affinity (Kd of 2800 pM) IL 2-R. These IL 2-R were functional, based on their ability to rapidly internalize IL 2. This model system will allow a detailed analysis of the mechanisms involved in the regulation of the immune response by IL 1 and IL 2.     

Functional status of interleukin 2 receptors expressed by immature (Lyt-2-/L3T4-) thymocytes

A subpopulation of phenotypically immature (Lyt-2-/L3T4-) thymocytes express receptors for the polypeptide hormone interleukin 2 (IL 2); however, these cells do not proliferate in vitro in response to IL 2. In investigating this phenomenon in greater detail, we observed that the IL 2 receptors (IL 2-R) on freshly isolated immature thymocytes bound IL 2 with about fivefold lower affinity (Kd approximately 100 pM) than IL 2-R on activated mature T cells and T cell lines (Kd approximately 20 pM). Furthermore, in contrast to activated T cells, Lyt-2-/L3T4- thymocytes did not endocytose bound IL 2. When stimulated in short-term culture with a combination of phorbol ester (PMA) and calcium ionophore, Lyt-2-/L3T4- thymocytes proliferated in a largely IL 2-dependent fashion. IL 2-R expression on these activated cells initially disappeared (at 24 hr) and subsequently reappeared (at 48 to 72 hr). Reexpressed IL 2-R on activated thymocytes resembled those on mature T lymphocytes in that they bound IL 2 with high affinity (Kd = 15 to 25 pM) and were capable of endocytosing IL 2. Taken together, these data place certain constraints on the putative physiologic role of IL 2 in intrathymic growth regulation.     

Inactivated Eastern Equine Encephalomyelitis Vaccines Prepared in Monolayer and Concentrated Suspension Chick Embryo Cultures

Eastern equine encephalomyelitis vaccines were prepared with virus propagated in stationary monolayer cultures and in concentrated suspension cultures of primary chick embryo cells. Virus pools for vaccine preparation were inactivated by three different methods: 0.05% formalin, 41 C heat, and 0.16% beta-propiolactone. Heat-and beta-propiolactone-inactivated vaccines maintained high hemagglutinating titers in the fluid state for at least 10 months, whereas formalin-inactivated vaccines lost their hemagglutinating activity within a few hours after treatment. The hemagglutinin of beta-propiolactone-inactivated virus particles was more dense than the hemagglutinin of the parent virus particles, as determined by sucrose density gradient centrifugation. The increase in density may be due to the degradation or removal of the lipid from the virus envelope. When administered to guinea pigs, all three vaccines stimulated hemagglutination-inhibiting, complement-fixing, and neutralizing antibodies and afforded protection against a live virus challenge. Test results showed that vaccines prepared with virus propagated in concentrated suspension cultures were more immunogenic and stimulated greater serologic responses in guinea pigs than vaccines derived from monolayer-propagated virus. The beta-propiolactone-inactivated vaccine was most protective, the heat-inactivated (41 C) vaccine next, and the formalin-inactivated vaccine least potent.     

Western equine encephalomyelitis vaccine produced in chick embryo cell cultures

A method was developed for production of a freeze-dried Western equine encephalomyelitis vaccine from virus propagated in chick embryo cell culture monolayers maintained with a serum-free medium. A sufficient concentration of virus accumulated in the cell culture fluids prior to the occurrence of viral cytopathology to permit the production of a vaccine relatively free from serum and cellular proteins. Inoculation with two mouse ld₅₀ doses of virus per 100 tissue culture cells was found to yield reproducible high virus titers at a convenient harvest time. These harvests were inactivated at 22 C by 0.05% formalin within 48 hr. Potency test results, as measured by the protection of immunized guinea pigs against an intracerebral virus challenge, indicated that the vaccine produced from the virus propagated in cell culture was equal in potency to a lot of whole chick embryo vaccine used to immunize laboratory and field workers subject to a high risk of infection.     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Isolation and identification of some guanidino compounds in the urine of patients with hyperargininaemia by liquid chromatography,thin-layer chromatography and gas chromatography-mass spectrometry

Liquid column chromatographic studies of monosubstituted guanidino compounds, which are excreted in the urine of patients with hyperargininaemia are reported. The guanidino-positive peaks, with the highest excretion values, were isolated from urine and the isolated compounds were identified by thin-layer chromatography and gas chromatography—mass spectrometry. Guanidinoacetic acid, N-α-acetylarginine, argininic acid, γ-guanidinobutyric acid, arginine and α-keto-δ-guanidinovaleric acid were found to be excreted at high levels in the urine of patients with hyperargininaemia compared with controls.     

Plasma exchange in the long-term management of Waldenstr?m's macroglobulinemia.

Two patients with Waldenström''s macroglobulinemia (WM), which had become resistant to cytotoxic drugs, were treated for features of the hyperviscosity syndrome by repeated plasma exchange with the continuous-flow blood-cell separator over periods of 36 and 28 months, respectively. After four initial weekly plasma exchanges the procedure was carried out every 4 to 6 weeks and both patients tolerated it well. Relative viscosity of the serum was maintained within the normal range in one patient, and both patients remained free of symptoms of the hyperviscosity syndrome. The results suggest that treatment of WM by long-term "maintenance" plasma exchange alone should be considered in any patient with complications due to chemotherapy or whose disease fails to respond to chemotherapy.     

Intercellular communication in the rat anterior pituitary gland: an in vivo and in vitro study

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.     

Tropical Splenomegaly Syndrome in Zambia: Further Observations and Effects of Cycloguanil and Proguanil

Nineteen Zambian patients with the tropical splenomegaly syndrome and sinusoidal lymphocytosis on liver biopsy were studied. The association of macrobulinaemia with the tropical splenomegaly syndrome has again been confirmed. Sixteen patients were treated with antimalarials—12 with cycloguanil pamoate alone, 3 with cycloguanil and proguanil, and 1 with proguanil alone. Twelve patients were observed for periods of sufficient length for the drug effect to be assessed, and in 11 there was a good response in terms of decrease in spleen size.Cycloguanil pamoate may be of value both for prophylaxis and treatment in areas where tropical splenomegaly syndrome is endemic.